Team:Wisconsin-Madison/experiments

From 2010.igem.org

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'''Background'''  
'''Background'''  
Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content.
Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content.
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'''Materials'''  
'''Materials'''  
*H2SO4/H2O (6:1 v/v)
*H2SO4/H2O (6:1 v/v)
*Cysteine hydrochloride
*Cysteine hydrochloride
*L-Frucose (calibration curve)
*L-Frucose (calibration curve)
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'''Cell Culture:''' (Final 50ml sample)
'''Cell Culture:''' (Final 50ml sample)
#Transform plasmids for high protein production
#Transform plasmids for high protein production
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#Induce with 1mM IPTG at OD600 = 0.05  
#Induce with 1mM IPTG at OD600 = 0.05  
#Grow cells to stationary phase
#Grow cells to stationary phase
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'''Protocol:'''
'''Protocol:'''
#Prepare overnight liquid culture of 50 ml sample
#Prepare overnight liquid culture of 50 ml sample
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#Centrifuge at 14,000g for 30 min at 4°C
#Centrifuge at 14,000g for 30 min at 4°C
#Dissolve pellet in 1 ml of sterile distilled water
#Dissolve pellet in 1 ml of sterile distilled water
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'''Quantification:''' Use negative controls of glucose and sterile distilled water
'''Quantification:''' Use negative controls of glucose and sterile distilled water
#Mix 4.5 ml of H2SO4/H2O (6:1 v/v) with 100uL of 1ml sample
#Mix 4.5 ml of H2SO4/H2O (6:1 v/v) with 100uL of 1ml sample
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#Measure absorbance at 396 nm and 427 nm
#Measure absorbance at 396 nm and 427 nm
#Difference in these measurements (after subtracted from pre-cysteine addition absorbance) can be directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml  
#Difference in these measurements (after subtracted from pre-cysteine addition absorbance) can be directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml  
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'''L-Fucose – Standard Curve'''
'''L-Fucose – Standard Curve'''
*Concentrations of L-Fucose ranging from 0 μg/ml to 100 μg/ml
*Concentrations of L-Fucose ranging from 0 μg/ml to 100 μg/ml
*L-Fucose was added to the appropriate amount of sterile distilled water to result in the following concentrations and 1ml of this preparation was taken from step 8(b) to 8(h) from Colonic Acid Quantification Assay (above)
*L-Fucose was added to the appropriate amount of sterile distilled water to result in the following concentrations and 1ml of this preparation was taken from step 8(b) to 8(h) from Colonic Acid Quantification Assay (above)
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[[Image:UW_LFucoseCurve|400px]]
[[Image:UW_LFucoseCurve|400px]]

Revision as of 03:22, 20 October 2010

Contents

Enzyme Treatment

Encapsulation

Colonic Acid Quantification

Parts used in this experiment
Part Number Function Expression Type Zip File
<partinfo>BBa_k318500</partinfo> Produces Trascription Factor RcsB Inducible - IPTG 500
<partinfo>BBa_k318501</partinfo> Produces Trascription Factor RcsA Inducible - IPTG 501
<partinfo>BBa_k318502</partinfo> Produces Trascription Factor RcsA & RcsB Inducible - IPTG 502
<partinfo>BBa_k200021</partinfo> Empty Vector/Contol Inducible - IPTG NA


Background Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content.
Materials

  • H2SO4/H2O (6:1 v/v)
  • Cysteine hydrochloride
  • L-Frucose (calibration curve)


Cell Culture: (Final 50ml sample)

  1. Transform plasmids for high protein production
  2. Incubate plate overnight (do not place in fridge before next step)
  3. Prepare liquid culture of each sample
  4. Induce with 1mM IPTG at OD600 = 0.05
  5. Grow cells to stationary phase


Protocol:

  1. Prepare overnight liquid culture of 50 ml sample
  2. Measure OD600
  3. To inactivate EPS-degrading enzymes and completely release EPS from cell surface:
    1. Boil sample for 15 min
    2. Cool to room temp
    3. Centrifuge at 14,000g for 30 min at 4°C
  4. Add three volumes of 70% ethanol to 40 ml of supernatant fraction
  5. Place in 4°C overnight
  6. Centrifuge at 14,000g for 30 min at 4°C
  7. Dissolve pellet in 1 ml of sterile distilled water


Quantification: Use negative controls of glucose and sterile distilled water

  1. Mix 4.5 ml of H2SO4/H2O (6:1 v/v) with 100uL of 1ml sample
  2. Heat mixture to 100°C for 20 min
  3. Cool to room temperature
  4. Measure absorbance at 396 nm and 427 nm
  5. Add 100 μL of 10M cysteine hydrochloride
  6. Measure absorbance at 396 nm and 427 nm
  7. Difference in these measurements (after subtracted from pre-cysteine addition absorbance) can be directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml


L-Fucose – Standard Curve

  • Concentrations of L-Fucose ranging from 0 μg/ml to 100 μg/ml
  • L-Fucose was added to the appropriate amount of sterile distilled water to result in the following concentrations and 1ml of this preparation was taken from step 8(b) to 8(h) from Colonic Acid Quantification Assay (above)


400px


See entire procedure : Download See original reference: http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link1,http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link2http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link3

Cell Survivability Testing

Parts used in this experiment
Part Number Function Expression Type Zip File
<partinfo>BBa_k318500</partinfo> Produces Trascription Factor RcsB Inducible - IPTG 500
<partinfo>BBa_k318501</partinfo> Produces Trascription Factor RcsA Inducible - IPTG 501
<partinfo>BBa_k318502</partinfo> Produces Trascription Factor RcsA & RcsB Inducible - IPTG 502

Best Combination


Inducible-Repressible Expression

Characterize pH Promoters

Parts Used: <partinfo>BBa_k318513</partinfo>

Amount of Regulators

IR-System - Arabinose

Parts Used: <partinfo>BBa_k318509</partinfo>, <partinfo>BBa_k318510</partinfo>, <partinfo>BBa_K318511</partinfo>, <partinfo>BBa_K318506</partinfo>

IR-System - pH

Parts Used: <partinfo>BBa_TUNED Part</partinfo>, <partinfo>BBa_K318506</partinfo>

IR-Lysis - pH

Parts Used: <partinfo>BBa_TUNED Part</partinfo>, <partinfo>BBa_K318507</partinfo>

Bile Induction

<partinfo>BBa_K318516</partinfo>, <partinfo>BBa_Lysis Part</partinfo> <partinfo>BBa_K318516</partinfo>, <partinfo>BBa_LYSIS Part</partinfo>

Encryption

Laboratory Notebooks

Media:Wisconsin-Madison2010_Notebook1.pdf

Media:Wisconsin-Madison2010_Notebook2.pdf

Media:Wisconsin-Madison2010_Notebook3.pdf

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